ABSTRACT:
Crystallization of enzymes in presence of impurities is important for
clarifying the role of enzymes in natural world. Although it is proposed that
impurities inhibit nucleation of enzyme crystallization, details are unclear.
In this study, crystallization of cellobiohydrolase from Aspergillus niger was
investigated by dynamic and time-resolved static
light scattering using cellobiose as an impurity. We aimed to clarify how
cellobiose inhibits cellobiohydrolase crystallization and to
crystallize cellobiohydrolase in concentrated cellobiose without using seeds.
The contribution of attractive forces to total intermolecular interactions of
cellobiohydrolase monomers increased with the molar ratio of
cellobiose/cellobiohydrolase (R(cb/ce)).
Association dynamics of cellobiohydrolase using lithium sulfate, however,
showed that the initial aggregation rate decreased with an increase in R(cb/ce). Because binding sites of
cellobioses to cellobiohydrolase molecules differed from those for the growth
of protein crystals, the binding of cellobioses would increase the chemical
potential of the cellobiohydrolase monomers, which gradually reduced supersaturation for growth as the aggregate size increased.
This result was in contrast with the conventional idea that cellobiose inhibits
the nucleation of cellobiohydrolase crystals. Gentle agitation of cellobiose-containing cellobiohydrolase solutions during sitting-drop
vapor-diffusion growth resulted in the growth of cellobiohydrolase single
crystals for all R(cb/ce)
conditions without using seeds.